布鲁氏菌BspF蛋白生物信息学分析及其对巨噬细胞炎症因子表达的影响  

Bioinformatics Analysis of Brucella BspF Protein and Its Effect on Expression of Inflammatory Factors in Macrophages

作　　者：孙灿 魏硕 鄢余静 赵天艺 朱德馨 邓兴梅 郭嘉 张辉[1,2,3] 孙志华 SUN Can;WEI Shuo;YAN Yujing;ZHAO Tianyi;ZHU Dexin;DENG Xingmei;GUO Jia;ZHANG Hui;SUN Zhihua(College of Animal Science and Technology,Shihezi University,Shihezi 832000,China;Collaborative Innovation Center for Healthy Sheep Breeding and Zoonotic Diseasa Prevention and Control,Shihezi 832000,China;Key Laboratory of Animal Disease Prevention and Control,Xinjiang Production and Construction Crops,Shihezi 832000,China)

机构地区：[1]石河子大学动物科技学院,石河子832000 [2]绵羊健康养殖与人兽共患病防控协同创新中心,石河子832000 [3]新疆生产建设兵团动物疾病防控重点实验室,石河子832000

出　　处：《中国畜牧兽医》2024年第5期1846-1856,共11页China Animal Husbandry & Veterinary Medicine

基　　金：石河子大学高层次人才项目(RCZK202038)。

摘　　要：【目的】对布鲁氏菌BspF蛋白进行生物信息学分析,并进一步探究BspF蛋白对巨噬细胞炎症因子表达的影响。【方法】利用生物信息学在线软件分析布鲁氏菌BspF蛋白的理化性质、亲/疏水性、信号肽、跨膜区、抗原决定簇及结构域、二级结构、三级结构。构建重组质粒pET-32a-BspF并将其转化大肠杆菌BL21(DE3)感受态细胞,用IPTG诱导表达BspF蛋白并进行纯化,通过SDS-PAGE和Western blotting检测蛋白表达及纯化情况。通过CCK-8法检测纯化后BspF蛋白对小鼠巨噬细胞(RAW264.7)的毒性,利用实时荧光定量PCR和Western blotting分别检测BspF蛋白对细胞中炎症相关因子mRNA和蛋白表达的影响。【结果】BspF蛋白分子质量为48.75 ku,属于不稳定蛋白,具有15个抗原决定簇,不存在跨膜区域和信号肽,二级结构以α-螺旋为主。成功构建了布鲁氏菌BspF基因原核表达载体,诱导表达后获得大小为60 ku的目标蛋白。纯化后蛋白以50μg/mL终浓度刺激RAW264.7细胞24 h后,与对照组相比,BspF组细胞中NLRP3、Caspase-1、IL-1β、IL-18的mRNA表达量均显著或极显著升高(P<0.05;P<0.01),NLRP3、Pro-Caspase-1蛋白表达量均极显著升高(P<0.01)。【结论】布鲁氏菌BspF蛋白能通过NLRP3炎症小体触发宿主细胞炎症反应,促进巨噬细胞炎症因子的表达。结果为布鲁氏菌致炎机制的研究和抗炎靶点的筛选提供理论基础。【Objective】The aim of this study was to analyze the bioinformatics of Brucella BspF protein,and further explore the effect of BspF protein on the expression of inflammatory cytokines in macrophages.【Method】The physical and chemical properties,hydrophilicity and hydrophobicity,signal peptide,transmembrane region,antigenic determinant and domain,secondary structure and tertiary structure of Brucella BspF protein were analyzed by bioinformatics online software.Constructed recombinant plasmid pET-32a-BspF and then transformed into Escherichia coli BL21(DE3)competent cells.BspF protein was induced by IPTG and purified.The expression and purification of BSPF protein were detected by SDS-PAGE and Western blotting.The toxicity of purified BspF protein on mouse macrophages(RAW264.7)was detected by CCK-8 method,and the effects of BspF protein on mRNA and protein expression of inflammatory factors in cells were detected by Real-time quantitative PCR and Western blotting,respectively.【Result】The results showed that BspF protein,with a molecular mass of 48.75 ku,was an unstable protein with 15 antigenic determinant,no transmembrane region and no signal peptide,and the secondary structure was dominated by alpha helix.The prokaryotic expression vector of Brucella BspF gene was successfully constructed,and the target protein with the size of 60 ku was obtained after induced expression.Compared with control group,after the purified protein was stimulated at the final concentration of 50μg/mL for 24 h,the mRNA expression of NLRP3,Caspase-1,IL-1βand IL-18 in BspF group were significantly or extremely significantly increased(P<0.05 or P<0.01),the protein expression of NLRP3 and Pro-Caspase-1 were extremely significantly increased(P<0.01).【Conclusion】Brucella BspF protein could trigger the inflammatory response of host cells through the NLRP3 inflammatome and promote the expression of inflammatory factors in macrophages,which provided a theoretical basis for the study of the inflammatogenic mechanism of Brucella and

关 键 词：布鲁氏菌 BspF蛋白 生物信息学 原核表达 炎性因子 

分 类 号：Q816[生物学—生物工程] S852.614[农业科学—基础兽医学]